Biocoat osteologic

Osteoclast differentiation assay was performed on hydroxyapatite disks (BD BioCoat Osteologic (BD Biosciences, San Diego, CA, USA) for 9 days. The media and all reagents were replaced every day to avoid the acidification of the medium. After culturing with cells, the hydroxyapatite discs were washed with 1 M NH₄OH to remove adherent cells. After rinsing with water, the hydroxyapatite discs were visually examined by light microscopy, and the area of resorption was calculated using the NIH Image J analysis software (http://rsbweb.nih.gov/ij/). The results were expressed as the percentage of the total area of the well that appears resorbed.

Cell proliferation on HA coated surfaces was measured using the WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium, monosodium salt] assay with Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). MC3T3-E1 cells were cultured in a non-osteogenic standard medium on untreated and plasma-treated HA-coated culture plates (BD BioCoat™ Osteologic™) at an initial density of 3 × 10³ cells per well. The non-osteogenic standard medium consisted of α Eagles’s minimal essential medium (αMEM, Gibco, ThermoFisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Lot No.002095, JRH Bioscience, Lenexa, KS, USA), and antibiotics (100 U/ml of penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml of amphotericin B; Sigma-Aldrich, St. Louis, MO, USA).
At 1, 2, and 4 day time points, 10 μL of the Cell Counting Kit solution was added into each well, followed by incubation of the culture plates at 37°C in 5% CO₂ for 2 hours. The absorbance was read at 450 nm with an automatic enzyme-linked immunosorbent assay reader (Multiskan Ascent MS353; Thermo Fisher Scientific).